a) Define the multistate test tube ensemble for CHA

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Tube On-Targets $(\Psi_{h}^{on})$ Off-Targets $(\Psi_{h}^{off})$
Step 0 {X,A,B,C,D} $\Psi_{0}^{L\leq L_{max}}$ - {X$\cdot$A, X$\cdot$B}
Step 1 {X$\cdot$A} {X,A} $\cup \Psi_{1}^{L\leq L_{max}}$
Step 2 {X$\cdot$A$\cdot$B} {X$\cdot$A, B} $\cup \Psi_{2}^{L\leq L_{max}}$
Step 3 {X$\cdot$A$\cdot$B$\cdot$C} {X$\cdot$A$\cdot$B, C} $\cup \Psi_{3}^{L\leq L_{max}}$
Step 4a {X$\cdot$A$\cdot$B$\cdot$C$\cdot$D} {X$\cdot$A$\cdot$B$\cdot$C, D} $\cup \Psi_{4a}^{L\leq L_{max}}$
Step 4b {X, A$\cdot$B$\cdot$C$\cdot$D} {A$\cdot$B$\cdot$C$\cdot$D, X} $\cup \Psi_{4b}^{L\leq L_{max}}$
Crosstalk $\cup_{n=1}${$\lambda^{reactive}$} $\Psi_{global}^{L\leq L_{max}}$ - $\cup_{n=1}${$\lambda^{cognate}$}

Reactants for system

Elementary step tubes for system:

Crosstalk tube

$ A \equiv A^{in} - A^{out} $

$ A^{toe} \equiv a $

$ A^{in} \equiv a-x-b-y $

$ A^{out} \equiv z*-c*-y*-b*-c* $

$ B \equiv B^{in} - B^{out} $

$ B^{toe} \equiv b $

$ B^{in} \equiv b-y-c-z $

$ B^{out} \equiv w*-d*-z*-c*-y* $

$ C \equiv C^{in} - C^{out} $

$ C^{toe} \equiv c $

$ C^{in} \equiv c-z-d-w $

$ C^{out} \equiv x*-a*-w*-d*-z* $

$ D \equiv D^{in} - D^{out} $

$ D^{toe} \equiv d $

$ D^{in} \equiv d-w-a-x $

$ D^{out} \equiv y*-b*-x*-a*-w* $

$ X \equiv y*-b*-x*-a* $

b) Multistate test tube design

c) Plots

The majority of the residual defect results from structural not concentration defects. However, in the crosstalk tube, the high concentration defect of strands C and D increase the ration of concentration defect to structural defect.